New Ochratoxin a Or Sclerotium Producing Species in Aspergillus Section Nigri

STUDIES IN MYCOLOGY 50: 45–61. 2004.

New ochratoxin A or sclerotium producing species in Aspergillus section Nigri

1* 1 1 2 Robert A. Samson , Jos A.M.P. Houbraken , Angelina F.A. Kuijpers , J. Mick Frank and Jens C. 3 Frisvad

1Centraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 AD Utrecht, the Netherlands; 233 Tor Road, Farnham, Surrey, GU9 7BY, U.K.; 3Center for Microbial Biotechnology, BioCentrum-DTU, Building 221, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark

*Correspondence: Robert A. Samson, [email protected]

Abstract: Aspergillus section Nigri includes some of the most important species for biotechnology and its species are of widespread occurrence. During our surveys of various food products and tropical soil we isolated several aspergilli belonging to section Nigri. In this paper, four new sclerotium and/or ochratoxin A producing species belonging to this section are proposed. In addition, based on a polyphasic approach using traditional characters, extrolites and β-tubulin sequencing, a provisional revision and synoptic key of section Nigri is proposed. Aspergillus costaricaensis was isolated from soil in Costa Rica and produces large pink to greyish brown sclerotia. Aspergillus lacticoffeatus was found on coffee beans in Venezuela and Indonesia, and is an effective producer of ochratoxin A. Aspergillus piperis was isolated from black ground pepper and produces large yellow to pink brown sclerotia. Aspergillus sclerotioniger was isolated from a green coffee bean and produces large yellow to red brown sclerotia and abundant ochratoxin A. The species A. homomorphus is validated. The ochratoxin A producing black aspergilli are revised. Fifteen species are provisionally accepted in Aspergillus section Nigri, four of these produce ochratoxin A. Ochratoxin A producing species of section Nigri occurring on grapes, raisins and in wine include A. carbonarius and to a lesser extent A. niger. Four species recovered from coffee, viz. A. carbonarius, A. niger, A. lacticoffeatus and A. sclerotioniger, all produce ochratoxin A, but other species of Nigri also occur on this substrate, including A. japonicus and A. tubingensis. The 10 species not producing ochratoxin A are especially interesting for biotechnological exploration, as many other extrolites are produced by these species.

Taxonomic novelties: Aspergillus costaricaensis Samson & Frisvad sp. nov., Aspergillus homomorphus Frisvad & Samson sp. nov., Aspergillus lacticoffeatus Samson & Frisvad sp. nov., Aspergillus piperis Samson & Frisvad sp. nov., Aspergillus sclerotioniger Steiman, Guiraud, Sage & Seigle-Mur. ex Samson & Frisvad sp. nov. Key words: Aspergillus niger, black aspergilli, ochratoxin A, pyranonigrin, sclerotia.

INTRODUCTION echinulate conidial ornamentations distinct from the remaining black Aspergillus taxa, which produce The black aspergilli are among the most common verrucose conidia. Within the verrucose category, A. fungi causing food spoilage and biodeterioration of fonsecaeus, A. acidus, A. niger var. niger, A. niger other materials. They have also been extensively used var. phoenicis, A. niger var. ficuum, A. niger var. for various biotechnological purposes, including tubingensis, A. niger var. pulverulentus, A. niger var. production of enzymes and organic acids (Schuster et awamori, A. citricus (A. foetidus) and A. citricus var. al. 2002). The taxonomy of Aspergillus section Nigri pallidus were recognized. has been studied by many taxonomists and was re- Aspergillus niger is the most frequently reported cently reviewed by Abarca et al. (2004). Mosseray species in this section, and has often been included in (1934) described 35 species black aspergilli, while biotechnological processes that are Generally Re- Raper and Fennell (1965) reduced the number of garded as Safe (GRAS). However, species concepts species accepted within their A. niger group to 12. Al- are uncertain in this complex and occasionally the Musallam (1980) revised the taxonomy of the A. niger name A. niger has been used for any member of the group, primarily based on morphological features. She section. Taxonomic studies using molecular methods recognized seven species (A. japonicus, A. carbon- have divided the A. niger complex into two species, A. arius, A. ellipticus, A. helicothrix, A. heteromorphus, niger and A. tubingensis (for overview see Abarca et A. foetidus, A. niger), and described A. niger as an al. 2004). Some further species have been described aggregate consisting of seven varieties and two for- but not considered in revisions or reviews. Aspergillus mae. Kozakiewicz (1989) distinguished A. ellipticus, ellipsoideus was described as a new species with A. heteromorphus, A. japonicus, A. helicothrix, A. ellipsoidal greyish black conidia (Rai & Chowdhery atroviolaceus and A. carbonarius as species exhibiting 1979). Aspergillus homomorphus and A. pseudohet-

45 SAMSON ET AL. eromorphus were invalidly described (no designated The names of colours are based on Kornerup & Wan- type, International Code of Botanical Nomenclature scher (1978). The cultures used for the molecular Art. 37) (Steiman et al. 1994; see Mouchacca 1999). study were grown in 2 mL malt peptone (MP) broth Recently, a new species A. vadensis, with a different using 10 % (v/v) of malt extract (Brix 10) and 0.1 % extrolite profile, colony characters and unusually low (w/v) bacto peptone (Difco) in 15 mL polystyrene citric acid production, was proposed (de Vries et al. centrifuge tubes. The cultures were incubated at 25 °C 2004a, b). without agitation for 7 d in light/darkness. The strains Ueno et al. (1991) were the first to report on ochra- selected included 1 to 8 representatives of the major toxin A (OA) production by a black Aspergillus taxa accepted by Al-Musallam (1980), Kozakiewicz species, A. foetidus. This was later confirmed by (1989) and Abarca et al. (2004) (see Table 1) in Téren et al. (1996) and Magnoli et al. (2003). Abarca addition to the new taxa described here and in de et al. (1994) reported that two strains of A. niger Vries et al. (2004b). produced OA, which was confirmed in numerous studies (Ono et al. 1995, Téren et al. 1996, 1997, Extrolite analysis Nakajima et al, 1997, Heenan et al. 1998, Accensi et Extrolites (includes secondary metabolites; for defini- al. 2001, Urbano et al. 2001, Dalcero et al. 2002, Da tion see Samson & Frisvad 2004) were analysed by Rocha et al. 2002, Abarca et al. 2003, Magnoli et al. HPLC using alkylphenone retention indices and diode 2003, Taniwaki et al. 2003, Suárez-Quiroz et al. array UV-VIS detection as described by Frisvad & 2004). Horie (1995) reported OA in A. carbonarius, Thrane (1987), with minor modifications as described which was confirmed by Wicklow et al. (1996), Téren by Smedsgaard (1997). Standards of ochratoxin A and et al. (1996), Heenan et al. (1998), Varga et al. B, aflavinine, asperazine, austdiol, kotanin and other (2000), Joosten et al. (2001), Da Rocha et al. (2002), extrolites from the collection at Biocentrum-DTU Cabanes et al. (2002), Sage et al. (2002), Abarca et al. were used to compare with the extrolites from the (2003), Battilani et al. (2003), Taniwaki et al. (2003), species under study. Pyranonigrin A was identified by Bellí et al. (2004) and Sage et al. (2004). Varga et al. comparison with literature UV and MS data (Hiort (2000) tested about 160 black Aspergillus strains from 2003, Hiort et al. 2004) collections and from field isolates for OA production using an immunochemical method and thin layer DNA Extraction, sequencing and analysis chromatography. The strains examined included 12 A. The total fungal genomic DNA was isolated using carbonarius and 45 A. japonicus strains from culture UltracleanTM Microbial DNA Isolation Kit (MoBio, collections and field isolates from all over the world, Solana Beach, U.S.A.) according to the manufac- including about 100 strains belonging to the A. niger turer’s instructions. Amplification of β-tubulin gene species complex. was mostly performed using the primers Bt2a and Ochratoxin A production was detected in about 6 Bt2b. Some strains in this study Bt-T10 (5'ACG ATA % of the strains from the A. niger species complex GGT TCA CCT CCA GAC 3') an Bt2b (Glass 1995). (Abarca et al. 1994, Téren et al. 1996). Of the 13 A. PCR was performed in a 25 µL reaction mixture carbonarius strains tested, six produced both OA and containing 1 µL of genomic DNA (10 ng/µL), 0.75 µL ochratoxins B (Fig. 8, Téren et al. 1996, Wicklow et of MgCl2 (50mM provided with BioTaq), 2.5 µL al. 1996). Aspergillus ellipticus, A. heteromorphus, A. Buffer with 10× NH4 (provided with BioTaq), 17.8 µL japonicus and A. tubingensis strains did not produce of ultra pure sterile water, 1.85 µL dNTP (1 mM), detectable amounts of ochratoxins. However, A. 0.50 µL of each primer (10 pmol/µL) and 0.1 µL japonicus was later claimed to produce OA (Dalcero BioTaq polymerase (5 U/µL, BiotaqTM DNA Poly- et al. 2002, Battilani et al. 2003). merase, Bioline Randolph, U.S.A.). Amplification was During our surveys of coffee, black pepper and performed in a GeneAmp PCR system 9700 (AB soil, several isolates of black aspergilli were recov- Applied Biosystems, Nieuwerkerk a/d IJssel, The ered. The purpose of this paper is to describe four new Netherlands); programmed for 5 min 94 °C followed species from section Nigri, distinguished from previ- by 35 cycles of 1 min denaturation at 94 °C followed ously known species by large sclerotia or unusual by primer annealing 1 at 55 °C and primer extension 1 conidial colours. Furthermore we wanted to suggest a min. at 72 °C and a final 7 min elongation step at 72 provisional revision of this industrially important °C. After amplification of the β-tubulin gene, excess section of Aspergillus based on a relatively small primers and dNTP’s were removed from the reaction number of typical strains of each taxon. mixture using a commercial GFX column, PCR DNA

Purification kit (Amersham Bioscience, Roosendaal,

MATERIALS AND METHODS The Netherlands). The purified PCR fragments were resuspended in 50 µL of TE buffer. The PCR fragments were directly sequenced in both directions with The methods and media for isolation and identifica- the primers Bt2a or BtT10 and Bt2b using a tion followed the procedures of Samson et al. (2004). 46 NEW SPECIES OF ASPERGILLUS SECTION NIGRI

Table 1. Cultures examined. Taxon name Strain number(s) Substratum and origin GenBank accession no. A. “aculeatus” CBS 620.78 = NRRL 2053 Unknown AY 585538 A. “aculeatus” CBS 114.80 Soil, India AY 585539 A. “brasiliensis” CBS 101740 = IMI 381727 Soil, Brazil AY 820006 A. aculeatus CBS 119.49 Unknown substratum, Indonesia AY 585541 CBS 172.66 = ATCC 16872 = IMI Tropical soil, unknown origin AY 585540 211388 = WB 5094 T A. carbonarius CBS 116.49 Unknown AY 819997 CBS 101697 = IBT 21854 External finga-coffee bean, Kenya AY 819994 CBS 126.49 = ATCC 10698 = IFO Unknown AY 819995 6648 = NRRL 363 (received as A. phoenicis) CBS 111.26 = ATCC 1025; = Paper, unknown origin AY 585532 ATHUM 2854 = CBS 556.65 = IMI 016136 = IMI 016136ii = LSHB Ac11 = MUCL 13583 = NCTC 1325 = NRRL 369 = NRRL 1987 = QM 331 = WB 369 NT A. costaricaensis CBS 115574 = CBS 23401 T Soil in Gaugin Garden on Taboga AY 820014 Island, Costa Rica A. ellipticus CBS 677.79 = IMI 278383 (Type of A. Sector in colony of Aspergillus AY 819993 helicothrix) ellipticus, CBS 482.65, Costa Rica A. ellipticus CBS 707.79 = IMI 278384 T Soil, Costa Rica AY 585530 A. flavus (outgroup) CBS 100927 = ATCC 16883 = CBS Cellophane, South Pacific Islands AY 819992 569.65 = IMI 124930 = LCP 89.2565 = WB 1957 A. foetidus CBS 564.65 =ATCC 16874 = IFO Unknown substratum, Japan AY 585533 4121 = IMI 104688 = IMI 104688ii = WB 4796 (Type of A. foetidus var. acidus) CBS 565.65 = ATCC 16884 = IFO Unknown substratum, Japan AY 585534 4123 = IMI 175963 = WB 4797 (Type of A. foetidus var. pallidus) A. heteromorphus CBS 117.55 = ATCC 12064 = IMI Culture contaminant of Trichophy- AY 585529 172288 = QM 6954 = WB 4747 T ton culture, Brazil A. homomorphus CBS 101889 T Soil of death sea area, Israel AY 820015 A. japonicus CBS 115.80 = IFO 5330 (Type of A. Unknown AY 820017 yezoensis) CBS 611.78 = NRRL 5118 Tropical soil, unknown origin AY 585544 CBS 113.48 = IMI 312983 = IMI Unknown AY 585531 016135ii = LSHB Ac44 = MUCL 13578 = NCTC 3792 = NRRL 4839 = WB 4839 (Type of A. atro-violaceus) CBS 568.65 = ATCC 16873 = IMI Soil, Panama AY 820018 211387 = NRRL 1782 = WB 1782 CBS 101.14 = IFO 4030 (received as Unknown AY 585543 A. atropurpureus) CBS 522.89 Air, the Netherlands AY 820019 CBS 114.51 T Unknown AY 585542 A. lacticoffeatus CBS 101884 Beans of Coffea arabica, Vene- AY 819999 zuela, Rubio district CBS 101886 = IBT 22032 Soil under Coffea robusta, AY 820003 Indonesia, Sumatra CBS 101883 T Surface sterilized beans Coffea AY 819998 robusta, Indonesia, South Sumatra A. niger CBS 101699 = IBT 6461 Foodstuff, unknown origin AY 585537 CBS 618.78 = IFO 739 = IMI 041871 Unknown AY 820004 = LSHB Ac72 = MUCL 28130 = NCTC 1692 = VTT D-71001 = NRRL 337 (believed to be Wehmer’s isolate of A. niger-citricus nom. nud., as A. foetidus) 47 SAMSON ET AL.

CBS 420.64 = ATCC 8740 = DSM Unknown AY 820002 872 = IMI 041875 = MUCL 30479 = NRRL 67 = NRRL 605 = NRRL 1737 = QM 330 = WB 67 (Isotype of A. fonsecaeus) CBS 101705 = IBT 18741 Carpet dust from school, Canada AY 820005 CBS 101698 = IBT 21853 Mesocarp finga - coffee bean, AY 820000 Kenya CBS 120.49 = ATCC 9029 = CECT Unknown substratum, U.S.A. AY 585535 2088 = DSM 2466 = IMI 041876 = MUCL 30480 = NRRL 3 = NRRL 566 = VKM F-3747 = VTT D-85240 = WB 3 = WB 566 (=’A. usamii’) CBS 557.65 = ATCC 16877 = IMI Unknown AY 820001 211394 = IOC 230 = WB 4948 (type of A. awamori) CBS 554.65 = ATCC 16888 = IFO Unknown AY 585536 33023 = IHEM 3415 = IMI 050566 = IMI 050566ii = JCM 10254 = NRRL 326 = WB 326 T A. piperis CBS 112811 = IBT 26239 T Grounded black pepper of tropical AY 820013 origin, Denmark A. pseudoheteromorphus CBS 101888 T Soil of death sea area, Israel AY 820016 A. sclerotioniger CBS 115572 = IBT 22905 T Green Arabica coffee, India, AY 819996 Karnataka A. tubingensis CBS 117.32 (received as A. ficuum) Unknown AY 820012 CBS 136.52 = ATCC 11362 = CBS Kuro-koji, Japan AY 820008 552.65 = IMI 211395 = WB 4757 (Type strain of A. saitoi; as A. phoenicis) CBS 425.65 = IAM 2170 (received as Unknown substratum, Japan AY 820009 A. pulverulentus) CBS 126.52 = WB 4860 = IFO 4115 Unknown AY 585528 (received as A. miyakoensis, identified as A. awamori) CBS 115657 = IBT 23434 Desert sand, Namibia AY 820011 CBS 161.79 = NRRL 4700 Unknown substratum, India AY 585527 CBS 134.48 = Biourge 726 = WB Unknown AY 820007 4875 T A. vadensis T CBS 113365 = IMI 313493 T Dead plant tissue, unknown origin AY 585531 T = ex-type culture.

DYEnamic ET Terminator Cycle Sequencing Kit 3700 Genetic Analyzer (AB Applied Biosystems, (Amersham Bioscience, Roosendaal, The Nether- Nieuwerkerk a/d IJssel, The Netherlands). A concen- lands). The sequence PCR reaction mixture, total sus was computed from the forward and reverse reaction mix is 10 µL, contained 1 µL of template sequences with software package Seqman and Editseq DNA (10–15 ng/µL), 4 µL Dye terminator RR mix, 4 from the lasergene package (DNAStar Inc., Madison, µL ultra pure sterile water and 1 µL primer Bt2a or WI). The alignments of the partial β-tubulin gene Bt2b (4 pmol/µL). The reaction was performed in a sequence data were performed using the software GeneAmp PCR system 9700 run in 9600 mode (AB package BioNumerics from Applied Maths and man- Applied Biosystems, Nieuwerkerk a/d Yssel, The ual adjustments for improvement were made by eye Netherlands); programmed for 25 cycles of 10 s where necessary. The phylogenetic analyses of se- denaturation at 96 °C followed by primer annealing 5 quence data were done using PAUP (Phylogenetic s at 50 °C and primer extension 4 min at 60 °C. Se- Analysis Using Parsimony) v. 4.0b10 (Swofford quencing products were purified according to the 2000). Alignment gaps were treated as fifth character manufacturer’s recommendations with Sephadex G-50 state, missing data were identified by ‘?’, uninforma- superfine column (Amersham Bioscience, Roosen- tive characters were excluded and all characters were daal, The Netherlands) in a multiscreen HV plate unordered and equal weight. Maximum parsimony (Millipore, Amsterdam, The Netherlands) and with analysis was performed for all data sets using the MicroAmp Optical 96-well reaction plate (AB Ap- heuristic search option. The robustness of the most plied Biosystems, Nieuwerkerk a/d IJssel, The Nether- parsimonious trees was evaluated by 1000 bootstrap lands). The samples were analyzed on an ABI PRISM replications (Hillis & Bull 1993). Other measures 48 NEW SPECIES OF ASPERGILLUS SECTION NIGRI including tree length, consistency index, retention toxin A, pyranonigrin A, orlandin (see Cutler et al. index and rescaled consistency index (CI, RI and RC) 1979), kotanin and desmethylkotanin. All eight strains were also calculated. of A. niger investigated produced pyranonigrin A and naphtho-γ-pyrones. CBS 101705, CBS 101698 and CBS 554.65 produced orlandin, kotanin and des- RESULTS methylkotanin and CBS 618.78, CBS 420.64, CBS 101705, and CBS 101698 produced ochratoxin A and All strains of the black aspergilli produced a large B. A. piperis CBS 112811 produced aurasperone B, number of known and as yet unknown extrolites. 14-epi-14-hydroxy-10,23-dihydro-24,25- Some of the most important extrolites are listed in dehydroaflavinine, and 10,23-dihydro-24,25- Table 1. Two strains of Aspergillus aculeatus (CBS dehydroaflavinine. Aspergillus sclerotioniger CBS 172.66 and CBS 119.49) produced secalonic acid D as 115572 produced pyranonigrin A, naphtho-γ-pyrones, earlier reported for this taxon (Andersen et al. 1977) ochratoxin A and B, and compounds related to fu- and in addition they both produced neoxaline. The nalenone or corymbiferan-lactones. All eight strains of latter metabolite was first reported from A. japonicus A. tubingensis produced asperazine, except CBS (Hirano et al. 1979, Konda et al. 1980), but we only 161.79. The latter strain produced tubingensin A and found neoxaline in A. aculeatus in this study. Two B (TePaske et al. 1989b, c), dihydrotubingensin A and other strains identified as A. aculeatus were quite B (Sings et al. 2001) and 14-epi-14-hydroxy-10,23- dissimilar to the two typical strains above: CBS dihydro-24,25-dehydroaflavinine, 10,23-dihy-dro- 620.78 produced secalonic acid D and some indole 24,25-dehydroaflavinine and 10,23-dihydro-24,25- compounds, while CBS 114.80 produced the same dehydro-21-oxo-aflavinine (TePaske et al. 1989a) indole compounds and okaramin H and I. Those indicating a difference between CBS 161.79 and other okaramins have earlier been reported from A. aculea- strains of A. tubingensis. All eight strains of A. tubin- tus (Hayashi et al. 1999). A. brasiliensis CBS 101740 gensis (Table 1) also produced pyranonigrin A and produced some naphtho-γ-pyrones including naphtho-γ-pyrones. Aspergillus vadensis CBS 113365 aurasperone B (Tanaka et al. 1972) and a series of produced nigragillin, asperazine, aurasperone B (a compounds that have not been structure elucidated naphtho-γ-pyrone) and a polar orlandin-like com- yet. pound. All four strains of A. carbonarius produced Among the isolates listed in Table 1, four species pyranonigrin A (earlier reported from A. niger, Hiort were able to produce OA. Ochratoxin A was consis- et al. 2004), ochratoxin A and naphtho-γ-pyrones. tently produced by A. carbonarius strains, in agree- Aspergillus costaricaensis produced trace amounts of ment with most other studies on this species (Abarca aurasperone B, pyranonigrin A, 14-epi-14-hydroxy- et al. 2004). Ochratoxin A was only produced by 10,23-dihydro-24,25-dehydroaflavinine, 10,23-dihy- some strains of A. niger sensu stricto, also in agree- dro-24,25-dehydroflavinine (those aflavinines were ment with numerous studies (Abarca et al. 2004). The found earlier in A. tubingensis CBS 161.79 = NRRL other producers of OA were the new species that are 4700, TePaske et al. 1989a), a funalenone-like com- described below, namely A. lacticoffeatus and A. pound (see Inokoshi et al. 1999) or a corymbiferan sclerotioniger. Both of these new species were iso- lactone-like compound (see Overy & Blunt 2004). A. lated from coffee. On the other hand OA production ellipticus CBS 677.79 and CBS 707.79 both produced by A. japonicus (Dalcero et al. 2002, Battilani et al. austdiol earlier reported from Aspergillus ustus (Vleg- 2003) was not confirmed. Similarly, no strains of A. gaar et al. 1974). Aspergillus foetidus CBS 564.65 and foetidus sensu stricto produced OA. The strain CBS CBS 565.65 both produced asperazine, earlier errone- 618.78 has been identified by different authors as A. ously reported from A. niger (Varoglou et al. 1997). foetidus, A. foetidus var. citricus or A. citricus and The strain examined by Varoglou et al. was actually produced OA (Téren et al. 1996). It was listed among an A. tubingensis (unpublished results, J.C. Frisvad). isolates of A. foetidus by Raper & Fennell (1965). Furthermore CBS 564.65 and CBS 565.65 produced Ochratoxin A production by this strain was confirmed antafumicin (Fujimoto et al. 1993). Both strains also here, but this strain has been shown to be A. niger and produced naphtho-γ-pyrones and pyranonigrin A. not A. foetidus (Kusters-van Someren et al. 1991, Aspergillus heteromorphus CBS 117.55 produced Parenicová et al. 1997, Accensi et al. 1999). several as yet unknown extrolites, including some Sclerotium production was not necessarily corre- indol-alkaloids. Aspergillus homomorphus CBS lated with OA production. It was suggested by Wick- 101889 and A. pseudoheteromorphus had identical low et al. (1996), that ochratoxin A was associated profiles of extrolites, including secalonic acid D. with sclerotium production of A. carbonarius. Asper- Aspergillus japonicus CBS 101.14, CBS 114.51 and gillus carbonarius occasionally produced sclerotia and CBS 522.89 did not produce any known extrolites. OA, but non-sclerotial strains of A. carbonarius could Aspergillus lacticoffeatus CBS 101886, CBS 101883, also produce ochratoxin A. A. tubingensis occasion- CBS 101884 and CBS 101885 all produced ochra- ally produces sclerotia but never produces ochratoxin 49 SAMSON ET AL.

A. No strains of A. niger have been found to produce DISCUSSION sclerotia yet, and other sclerotium producers, such as A. ellipticus, A. aculeatus, A. costaricaensis, and A. Approximately 108 taxa (species, subspecies and piperis also did not produce OA. Aspergillus scle- varieties) have been described in Aspergillus section rotioniger, however, produced abundant sclerotia and Nigri (Mosseray 1934, Raper & Fennell 1965, Samson OA. 1979, 1992, Al-Musallam 1980, Kozakiewicz 1989). Maximum parsimony analysis of the sequence data Of these, we provisionally accept 15 taxonomic enti- was restricted to 5000 equally most parsimonious ties, including the four new species described here. trees (TL = 719 steps, CI= 0.701 RI = 0.898, RC = The reason for the multiplicity of proposed names 0.630), one of which is shown in Fig. 1. The tree was may be that isolates of section Nigri are readily iso- rooted using A. flavus. This species was chosen after lated world-wide but are difficult to distinguish. examining the results of Peterson (2000). The boot- Often very small differences in texture or conidial strap support, based on fast stepwise addition, from colour have been used as the basis for distinguishing 1000 replicates is shown at the nodes. The cladogram new taxa. Phenotypic comparisons of a broad collec- indicates that there are five major clades in section tion of black aspergilli showed that 15 taxa can be Nigri. The first clade contains A. heteromorphus and distinguished. Most of these species could be distin- A. ellipticus, but these two species are clearly very guished by combinations of colony and micromor- distantly related. The next clade contains A. carbon- phological characters and extrolite profiles, including arius and A. sclerotioniger and this clade is a sister the new species described below in addition to A. clade to a major clade containing species usually ellipticus, A. carbonarius, A. japonicus, A. vadensis, included in the A. niger complex (Al-Musallam 1979), and A. heteromorphus. However, A. niger, A. tubin- but it also includes the two new species A. piperis and gensis and A. foetidus remain difficult to differentiate A. costaricaensis. This major clade includes two using phenotypic methods. These taxa can be differen- subclades, one with A. niger and A. lacticoffeatus, and tiated by DNA sequences of the cytochrome b gene one with A. vadensis, A. tubingensis, A. foetidus, A. (Yokoyama et al. 2001), ITS (Parenicová et al. 2001) piperis and A. costaricaensis. The next clade includes and β-tubulin (De Vries et al. 2004b) and by RFLP A. homomorphus and the closely related A. pseudohet- and other fingerprinting methods (Abarca et al. 2004). eromorphus and two strains identified as A. aculeatus. No phenotypic methods have yet been found that can This is the only clade containing both uniseriate and distinguish between A. niger, A. foetidus and A. tubin- biseriate aspergilli. The last clade includes the two gensis, except that they can be differentiated based on common uniseriate species A. aculeatus sensu stricto production of the extrolites asperazine, antafumicins and A. japonicus. This analysis, along with the pheno- and ochratoxin A and/or orlandins (Table 2). However typic data, supports our recognition of the newly old deteriorated strains sometimes do not produce any described species. We also conclude that CBS 101740 of these compounds and then molecular methods (“A. brasiliensis”) represents a new species distinct would be necessary to distinguish them. from all the other species of Aspergillus, and this is The β-tubulin nucleotide sequence cladogram (Fig. supported by 70 % majority-rule consensus analyses 1) is divided into four clades with no obvious sister but with a low bootstrap value (51 %). This species is group relationships, thus it is not possible to infer any represented by several isolates (Varga et al. 2000) and deeper phylogenetic relationships between these will be described elsewhere. Aspergillus piperis has groups. Within the four clades the phylogenetic struc- similar sequences to A. foetidus and differs only by six ture is more resolved. In the first clade, two very base pair changes; however it is supported by consen- unique species, A. heteromorphus and A. ellipticus, sus and bootstrap (85 %). Aspergillus costaricaensis is appear to be distantly related. The discovery of more a new species supported by consensus but with a poor taxa in section Nigri or the use of more than one gene bootstrap value (53 %). Aspergillus sclerotioniger is a for constructing the cladogram may help resolve this new species supported by consensus and bootstrap relationship. Aspergillus heteromorphus and A. ellipti- (100 %). All the three strains of A. lacticoffeatus have cus are also phenotypically very different. The next identical sequences to the eight strains of A. niger major clade consists of A. carbonarius, the A. niger studied. The strains of A. lacticoffeatus have strikingly complex and the four new species. Most of these different in colony colour and morphology and also species produce pyranonigrin A and naphtha-γ- have a different extrolite pattern. Multilocus DNA pyrones. sequences might reveal genetic differences between A. lacticoffeatus and A. niger. CBS 101888 and 101889, the ex-type strains of Aspergillus homomorphus and A. heteromorphus, had identical sequences.

50 NEW SPECIES OF ASPERGILLUS SECTION NIGRI

Table 2. Production of sclerotia, ochratoxin A and other extrolites by species in Aspergillus section Nigri. Species Ochratoxin A Sclerotia Pyranonigrin N-γ-P1 Asp2 SeD3 Ant4 Afl5 Cor6 Kot7 A. aculeatus – +/– – – – + – – – – A. brasiliensis – – – + – – – – – – A. carbonarius + +/– + + – – – – – – A. costaricaensis – + – + – – – + + – A. ellipticus – + – – – – – – – – A. foetidus – – + + + – + – – – A. heteromorphus – – – – – – – – – – A. homomorphus8 – – – – – + – – – – A. japonicus – – – – – – – – – – A. lacticoffeatus + – + – – – – – – + A. niger +/– – + + – – – – – +/– A. piperis – + + + – – – + – – A. sclerotioniger + + + + – – – – + – A. tubingensis – +/– + + + – – – – – A. vadensis – - – + + – – – – – 1N-γ-P: Naphtho-γ-pyrones; 2Asp = asperazine; 3SeD = secalonic acid D; 4Ant = antafumicin; 5Afl = aflavinines; 6Cor = Corymbiferan lactones; 7Kot = Kotanins (kotanin, desmethylkotanin, orlandin); 8A. pseudoheteromorphus was not different from A. homomorphus, but none of the species have been validly described.

88 Aspergillus heteromorphus CBS 117.55 T 100 Aspergillus ellipticus CBS 707.79 T Aspergillus ellipticus CBS 677.79 Aspergillus carbonarius CBS 101697 54 Aspergillus carbonarius CBS111.26 NT 93 Aspergillus carbonarius CBS 116.49 100 Aspergillus carbonarius CBS 126.49 Aspergillus sclerotioniger CBS 115572 T Aspergillus lacticoffeatus CBS 101883 T Aspergillus lacticoffeatus CBS 101884 Aspergillus niger CBS 101698 Aspergillus niger CBS 557.65 Aspergillus niger CBS 420.64 58 100 Aspergillus niger CBS 101705 Aspergillus niger CBS 101699 51 Aspergillus lacticoffeatus CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis CBS 101740 90 Aspergillus vadensis CBS 113365 T Aspergillus tubingensis CBS 134.48 T Aspergillus tubingensis CBS136.52 Aspergillus tubingensis CBS 425.65 80 Aspergillus tubingensis CBS 126.52 Aspergillus tubingensis CBS 161.79 Aspergillus tubingensis CBS 115657 Aspergillus tubingensis CBS 117.32 93 Aspergillus foetidus CBS 564.65 T 85 Aspergillus foetidus CBS 565.65 53 Aspergillus piperis CBS 112811 T Aspergillus costaricaensis CBS 115574 T 100 Aspergillus homomorphus CBS 101889 92 Aspergillus homomorphus CBS 101888 99 Aspergillus "aculeatus” CBS 620.78 Aspergillus "aculeatus” CBS 114.80 78 Aspergillus aculeatus CBS 119.49 Aspergillus aculeatus CBS 172.66 T 100 Aspergillus japonicus CBS 115.80 Aspergillus japonicus CBS 113.48 Aspergillus japonicus CBS 568.65 Aspergillus japonicus CBS 611.78 100 Aspergillus japonicus CBS 101.14 Aspergillus japonicus CBS 522.89 Aspergillus japonicus CBS 114.51 T Aspergillus flavus CBS 100927 NT 10 changes Fig. 1. One of the 5000 equally MPT of 719 steps based on heuristic search partial β-tubulin sequences with A. flavus as an outgroup. The branches in bold are 100 % in the 70 % majority-rule consensus of equally parsimonious trees. The numbers represent bootstrap percentages > 50 % (CI = 0.701, RI = 0.898 RC = 0.630, HI = 0.299). Names in blue are ochratoxin producing taxa. Taxa in red contain isolates which can produce ochratoxin.

51 SAMSON ET AL.

The first subclade, sister group to the A. niger com- Taxonomy plex, consists of species with large conidia and ability to produce sclerotia and ochratoxin A: A. carbonarius Aspergillus costaricaensis Samson & Frisvad sp. and A. sclerotioniger. The two latter species also share nov. MycoBank MB500007. the slow groth at 37 ºC. These species share the ochratoxin A production with A. niger and A. lacticoffeatus Aspergillo nigro similis, capitulis biseriatis, sed sclerotiis in the next subclade and the ability to produce scle- roseis vel grisello-luteis et vesiculis metulisque majoribus rotia with A. tubingensis, A. foetidus, A. piperis and A. differens. Typus CBS H-13437 costaricaensis in the last subclade. Sclerotium production in A. tubingensis and A. foetidus is rare, however. Type: CBS 115574 = IBT 23401, ex soil in Gaugin Garden In the next subclade A. niger sensu stricto and A. on Taboga Island, Costa Rica, Martha Christensen, Nov. lacticoffeatus cannot be separated based on their β- 2000. tubulin sequences. In agreement with this, they share the ability to produce OA, pyranonigrin, kotanins and Colony diameters at 7 d 25 °C, in mm: CYA: 63–78 in not having the ability to produce sclerotia. Again, mm, MEA 26–62 mm, YES: 77–80 mm, OAT: 41–56 there are several differences, including lack of naph- mm, CREA: 38–50 mm, thin colonies with poor tha-γ-pyrones in A. lacticoffeatus, the sulphur yellow sporulation, strong acid production, CYA at 37 ºC: mycelium on YES agar and the smooth to finely 58–62 mm. Colony colours and texture. On CYA25 roughened light brown to dark blonde conidia of A. and MEA only a few conidiophores are produced, lacticoffeatus. The third subclade consists of A. conidial areas are black; mycelium white, inconspicu- vadensis, A. tubingensis, A. foetidus, A. piperis and A. ous; sclerotia abundantly present, large (1.2–1.8 mm), costaricaensis. The three first species are united by subglobose to ellipsoidal, pink to grayish yellow. production of naphtho-γ-pyrones and asperazine, Reverse on CYA pale yellow, on MEA medium– while the latter two species produce naphtho-γ- yellow. Conidial heads radiate, splitting into 5–8 pyrones and aflavinins. The large central clade con- defined columns, stipes (800–)1000–1700(–1900) × sisting of A. carbonarius, A. sclerotioniger, A. niger, (12–)13–20(–22) µm, walls thick, smooth, hyaline; A. lacticoffeatus, A. brasiliensis, A. vadensis, A. vesicles large (40–)45–70(–90) µm wide, globose; tubingensis, A. foetidus, A. piperis and A. costaricaen- biseriate; metulae covering entire vesicle, measuring sis appears to be monophyletic and all species share 30–60 × 3–4 (at base) to 8–11 µm (at top); phialides the ability to produce naphtho-γ-pyrones and 7–9.5 × 3–5 µm; conidia globose to subglobose, (3.1–) pyranonigrin A, except that the naphtho-γ-pyrones has 3.5–4.3(–4.5), smooth when young, becoming distinct been lost in A. lacticoffeatus and pyranonigrin A has rough walled, dark brown. been lost in A. vadensis. The last two clades include uniseriate species and the biseriate A. homomorphus. Extrolites: Aurasperone B and pyranonigrin A, 14-epi- On the other hand all isolates of A. aculeatus and A. 14-hydroxy-10,23-dihydro-24,25-dehydro-aflavinine, homomorphus share the production of secalonic acid and 10,23-dihydro-24,25-dehydro-aflavi-nine, fu- D, not found in any other black Aspergillus species. nalenone-like compound similar to the corymbiferan Unexpectedly the distinction between uniseriate and lactones (see A. sclerotioniger). biseriate species is only partly supported by nucleotide sequence data. Originally the two types of black Distinguishing features: This species is characterised aspergilli were distinctly separated (Peterson 2000, by its pink to greyish yellow sclerotia and large vesi- Varga et al. 2000, Parenicová et al. 2001). The separa- cles and metulae. tion of A. aculeatus sensu lato into two separate clades indicate that more than one species may exist. The Aspergillus lacticoffeatus Frisvad & Samson sp. group of uniseriate black aspergilli should be further nov. MycoBank MB500008. examined before taxonomic conclusions for that group are drawn. However, our examination of the type Aspergillo nigro similis, capitulis biseriatis, sed coloniis isolates of Aspergillus homomorphus and A. pseudo- dilute brunneis et vesiculis metulisque majoribus et conidiis heteromorphus show that these species are identical. asperellis differens. Typus CBS H-13436 Both species were invalidly described and below we validate the species and name it A. homomorphus. Type: CBS 101883 = IBT 22031 ex surface disinfected In combination with the phenotypc and extrolite green robusta coffee bean in coffee farm, Labu Kompong of characters the β-tubulin sequences revealed the dis- Ngarip Village, Ulu Belu territory, Lampung highlands of tinction of the 15 taxa incluing four new species. southern Sumatra, Indonesia, J.M. Frank. However a multigen sequence approach will be necessary to get a better insight in the species complexes of A. niger/tubingensis and A. aculeatus/A. japonicus.

52 NEW SPECIES OF ASPERGILLUS SECTION NIGRI

Fig. 2. Aspergillus costaricaensis. Seven-day-old cultures on A. CYA and B. MEA. C. Conidiophore. D. Detail of a conidiophore showing large metulae. E. Detail of a 7-day-old colony showing sclerotia. F. Conidia. G. Scanning electron micrograph photo of conidia. Scale bars: C, D, F = 10 µm, E = 1 mm, G = 1 µm. 53 SAMSON ET AL.

Fig. 3. Aspergillus lacticoffeatus. Seven-day-old cultures on A. CYA and B. MEA. C, D. Conidiophores. E. Conidia. F. Scan- ning electron micrograph photos of conidia. Scale bars:: C–E = 10 µm, F = 1 µm.

54 NEW SPECIES OF ASPERGILLUS SECTION NIGRI

Fig 4. Aspergillus piperis. Seven-day-old cultures on A. CYA and B. MEA. C, D. Conidiophores. E. Conidia. F. Scanning electron micrograph photo of conidia. Scale bars: C–E = 10 µm, F = 1 µm.

55 SAMSON ET AL.

Fig. 5. Aspergillus sclerotioniger. Seven-day-old cultures on A. CYA and B. MEA. C. Conidiophore. D. Conidial head. E. detail of a 7-day-old colony showing sclerotia. F. Conidia. G. Scanning electron micrograph photo of conidia. Scale bars: C–D, F–G = 10 µm, E = 2 mm.

56 NEW SPECIES OF ASPERGILLUS SECTION NIGRI

Other strains: CBS 101885 = IBT 22029, ex surface disin- many aspergilla are produced on all media; hyphae fected ripe green arabica coffee bean, farm Agua Blanco, inconspicuous, white; large sclerotia (1-17 mm) Rubio district, Venezuela, J.M. Frank; CBS 101884 = IBT abundantly produced on all media, white when young 22030, ex surface disinfected ripe green arabica coffee becoming yellow to pink brown at age; exudate pre- bean, farm Agua Blanco, Rubio district, Venezuela, J.M. sent like small hyaline droplets; reverse uncoloured, Frank; CBS 101886 = IBT 22032, ex soil under robusta cherry coffee of a compacted soil drying yard, Karangsari, pale to creamy. Conidial heads radiate; stipes (300–) Pulo Pannggung subdistrict, Sumatra, Indonesia, J.M. 400–3000 × (7–)12–15(–20) µm, walls thick, smooth, Frank hyaline; vesicles (40–)45–50(–55) µm wide, nearly spherical; biseriate; metulae covering virtually the Colony diameters at 7 d 25 °C, in mm: CYA: 71–76 entire surface of the vesicle, measuring (20–)25–30(– mm, MEA 52–70 mm, YES: 75–80 mm, OAT: 32–36 35) × 3–6 µm; phialides (5.5–)6–7.5(–8) × 3–4 µm; mm, CREA: 32–44 mm, thin colonies with poor conidia subglobose to broadly ellipsoidal, 2.8–3.6 × sporulation, strong acid production, CYA at 37 ºC: 2.8–3.4 µm, smooth when young to very rough with 59–75 mm. Colony colours and texture. Conidial irregular bars/striations. areas first white then becoming hair brown (5E4) to dark blonde (5D4) and densely packed on CYA25, Extrolites: Aurasperone B, 14-epi-14-hydroxy-10,23- hyphae usually inconspicuous, no sclerotia on any dihydro-24,25-dehydroaflavinine, and 10,23-dihydro- medium, no exudates present, reverse cream to light 24,25-dehydroaflavinine. brown on CYA, colony granular, sometimes sulcate. The conidial heads are globose at first and later occa- Distinguishing features: This species is characterized sionally developing into several conidial columns on by its yellow to pink brown sclerotia, subglobose to each head. Colonies on CZ similar as on CYA, only broadly ellipsoidal and distinctly roughened conidia. reverse is uncoloured on CZ. Growth on YES is characterized by sulfur yellow mycelium formation. Aspergillus sclerotioniger Samson & Frisvad sp. × Conidial heads radiate; stipes short (200–)300–1200 nov. MycoBank MB500010. (7–)10–15(–18) µm, walls thick, smooth, orange– brown; vesicles (40–)45–60(–65) µm wide, nearly Aspergillo carbonario similis, capitulis biseriatis, sed spherical; biseriate; metulae covering virtually the mycelio luteo, sclerotiis luteis vel aurantiacis vel rubro- entire surface of the vesicle, measuring 12–25 × 3–6 brunneis, hyphis spicularibus luteis in agaro YES formatis µm; phialides 7–10 × 3–4 µm; conidia subglobose, et conidiis majoribus differens. Typus CBS H-13433. 3.5–4.1 × 3.4–3.9 µm, usually smooth to very finely roughened. No sclerotia observed Type: CBS 115572 = IBT 22905 ex surface disinfected green Arabica coffee bean, Karnataka, India, J.M. Frank. Extrolites: Ochratoxin A, ochratoxin B, pyranonigrin A, orlandin, kotanin. Colony diameters at 7 d 25 °C, in mm: CYA: 71–78 mm, MEA 60–72 mm, YES: 72–80 mm, OAT: 42–56 Distinguishing features: This species is characterized mm, CREA: 19–25 mm, thin colonies with poor by its hair brown to dark blonde colonies, biseriate sporulation, strong acid production, CYA at 37 ºC: 7– conidial heads with large vesicles and smooth to very 16 mm. Colony colours and texture. On CYA25 and finely roughened conidia. MEA only a few conidiophores are produced, conidial areas are black; mycelium yellow, conspicuous; Aspergillus piperis Samson & Frisvad sp. nov. sclerotia abundantly present, large (1–1.6 mm), (sub)globose, yellow to orange to red brown covered MycoBank MB500009. by yellow mycelium. Reverse on CYA pale, on MEA medium–yellow. Conidial heads radiate; stipes short Aspergillo nigro similis, capitulis biseriatis, sed sclerotiis × luteis vel roseo-brunneis et conidiis subglobosis vel late (400–)500–800(–1200) (12–)14–16(–18) µm, walls ellipsoideis distincte asperatis differens. Typus CBS H- thick, smooth, hyaline; vesicles (30–)35–45(–50) µm 13434. wide, pyriform; biseriate; metulae covering three quarters of the vesicle, measuring 8–14 × 4–6 µm; Type: CBS 112811 = IBT 26239, ex grounded black pepper phialides 6.5–9.5 × 3–5 µm; conidia subglobose, of tropical origin, Kgs. Lyngby, Denmark, K.F. Nielsen. (4.7–)5–6(–6.4) × (4.5–)4.9–5.6(–6.1) µm, smooth when young, becoming verruculose, dark brown. Colony diameters at 7 d 25 °C, in mm: CYA: 60–75 mm, MEA 59–78 mm, YES: 79–83 mm, OAT: 45–54 Extrolites: Ochratoxin A, ochratoxin B, traces of mm, CREA: 43–48 mm, thin colonies with poor aurasperone B, and pyranonigrin A. The isolates sporulation, strong acid production, CYA at 37 ºC: produce a compound with a chromophore like that of 64–82 mm. Colony colours and texture. Conidial the corymbiferans produced by Penicillium hordei areas black and sparsely produced, after sub-culturing 57 SAMSON ET AL.

Stolk (Overy & Blunt 2004). A compound with a 14. A. tubingensis chromophore close to these compounds is funalenone, 15. A. vadensis isolated from a fungus identified as A. niger (Inokoshi et al. 1999). This funalenone-like extrolite is also Conidia more than 6 µm diam: 3, (5) produced by A. costaricaensis, but has not been found Conidia spinulose: (3), 5, 6, 8, 9 in any strain of A. niger or A. tubingensis. Conidia strongly ellipsoidal: (1), 5, (6) Metulae not produced: 1, 6 Distinguishing features: This species is characterized Metulae less than 15 µm in length: (7), (8), 9, (10), yellow mycelium, yellow to orange to red brown (11), 13, (14), (15) sclerotia, yellow spicular hyphae on YES agar and Production of sclerotia: (1), (3), 4, (5), (6), 12, 13, large conidia. This species is related to Aspergillus (14) carbonarius. Sclerotia yellow to orange: 13 Sclerotia yellow to pinkish brown: 12 Aspergillus homomorphus Steiman, Guiraud, Sclerotia pint to grayish yellow: 3 Sage & Seigle-Mur. ex Samson & Frisvad, sp. Colony diameter at 25 ºC on CYA, 7 d, less than 30 nov. MycoBank MB500011. mm: 15 Colony diameter at 37 ºC on CYA, 7 d, larger than 70

mm: 2, 7, 10, 11, 12, 14 Latin description: Systematic and Applied Microbiol- Colony diameter at 37 ºC on CYA 7.d, between 55 ogy 17(4): 621. 1995. and 65 mm: 4, 15 Colony diameter at 37 ºC on CYA 7 d, less than 40 = Aspergillus homomorphus Steiman, Guiraud, Sage & mm: 1, 3, 5, 6, 8, 9, 13 Seigle-Mur., Systematic and Applied Microbiology 17(4): 621. 1995. [Nom.inval., Art. 37.4.] Colony diameter at 37 ºC on CYA, 7 d, 0 mm: (5), 8 = Aspergillus pseudo-heteromorphus Steiman, Guiraud, Acid production on CREA agar weak or not present: Sage & Seigle-Mur., Systematic and Applied Micro- (1), (7), 8, 9 biology 17(4): 622. 1995. [Nom.inval., Art. 37.4.] Conidium colour en masse light brown to dark blonde: 10, 15 Type: CBS 101889, soil of death sea area, Israel. Conidium colour en masse greenish–olive: 8, (15) Production of ochratoxin A: 3, 10, (11), 13 Both species were described without designating a Production of pyranonigrin A: 3, 7, 10, 11, 12, 13, 14 holotype specimen. Both taxa are identical and we are Production of one or more naphtha-γ-pyrones: 2, 3, 4, validating the name by depositing herb. CBS 101889 7, 11, 12, 13, 14, 15 as holotype. Production of asperazine: 7, 14, 15 Production of secalonic acid D: 1, 9 Extrolites: secalonic acid. Production of aflavinines: 4, 12, (14) Production of antafumicins: 7 Production of corymbiferan lactone/funalenone-like Distinguishing features: Short metulae, echinate compounds: 4, 13 conidia (spines up to 1.5 µm), secalonic acid D Production of kotanin, desmethylkotanin and/or orlandin: 10, (11) Production of austdiol: 5 Provisional synoptic key to species in Aspergillus Production of neoxaline: (1) section Nigri (Numbers in parentheses: feature not always present) Species list: 1. A. aculeatus 2. A. brasiliensis ined ACKNOWLEDGEMENTS 3. A. carbonarius

4. A. costaricaensis We thank Martha Christensen for donating some of the 5. A. ellipticus cultures studied and Kristian Fog Nielsen for analyzing A. 6. A. japonicus piperis chemically. The research was supported by the 7. A. foetidus Danish Technical Research Council (Program for Predictive 8. A. heteromorphus Biotechnology) and the Center for Advanced Food Studies 9. A. homomorphus (LMC). Walter Gams kindly prepared the Latin diagnoses. 10. A. lacticoffeatus 11. A. niger 12. A. piperis 13. A. sclerotioniger

58 NEW SPECIES OF ASPERGILLUS SECTION NIGRI

REFERENCES and detection using coconut cream agar. Journal of Food Mycology 1: 67–72. Abarca ML, Accensi F, Bragulat MR, Castella G, Cabañes Hiort J (2003). Neue Naturstoffe aus Schwam-assozierten FJ (2003). Aspergillus carbonarius as the main source Pilzen des Mittelmeeres. Matematisch- of ochratoxin A contamination in dried wine fruits from Naturwissenschaftlichen Fakultät der Heinrich-Heine- the Spanish market, Journal of Food Protection 66: Universität, Düsseldorf, Germany. 504–506. Hiort J, Maksimenka K, Reichert M, Perovic-Ottstadt S, Abarca ML, Accensi F, Cano J, Cabañes FJ (2004). Taxon- Lin WH, Wray V, Steube K, Schauman K, Weber H, omy and significance of black aspergilli. Antonie van Proksch P, Ebel R, Muller, WEG, Bringmann G (2004). Leeuwenhoek 86: 33–49. New natural products from the sponge-derived fungus Abarca ML, Bragulat MR, Castella G, Cabañes FJ (1994). Aspergillus niger. Journal of Natural Products 67: Ochratoxin A production by strains of Aspergillus niger 1532–1543. var. niger. Applied and Environmental Microbiology 60: Hirano A, Iwai Y, Masuma R, Tei K, Ōmura S (1979). 2650–2652. Neoxaline, a new alkaloid produced by Aspergillus ja- Accensi F, Abarca ML, Cano J, Figuera L, Cabañes FJ ponicus. Production, isolation and properties. Journal of (2001). Distribution of ochratoxin A producing strains Antibiotics 32: 781–785. in the A. niger aggregate. Antonie van Leeuwenhoek 79: Horie Y (1995). Productivity of ochratoxin A of Aspergillus 365–370. carbonarius in Aspergillus section Nigri. Nippon Kin- Al-Musallam A (1980). Revision of the black Aspergillus gakkai Kaiho 36: 73–76. species. Ph.d. thesis. Rijksuniversiteit Utrecht, Utrecht. Inokoshi J, Shiomi K, Masuma R, Tanaka H, Yamada H, Andersen R, Büchi G, Kobbe B, Demain AL (1977). Ōmura S (1999). Funalenone, a novel collagenase in- Secalonic acids D and F are toxic metabolites of Asper- hibitor produced by Aspergillus niger. Journal of Anti- gillus aculeatus. Journal of Organic Chemistry 42: 352– biotics 52: 1095–1100. 353. Joosten HMLJ, Goetz J, Pittet A, Schellenberg M, Bucheli Battilani P, Pietri A, Bertuzzi AT, Languasco L, Giorni P, P (2001). Production of ochratoxin A by Aspergillus Kozakiewicz Z (2003). Occurrence of ochratoxin A- carbonarius on coffee cherries. International Journal of producing fungi in grapes grown in Italy. Journal of Food Microbiology 65: 39–44. Food Protection 66: 633–636. Konda Y, Onda M, Hirano A, Ōmura S (1980). Oxaline and Bellí N, Pardo E, Marín S, Farré G, Ramo RJ, Sanchis V neoxaline. Chemical and Pharmaceutical Bulletin 28: (2004.) Occurrence of ochratoxin A and toxigenic po- 2987–2993. tential of fungal isolates from Spanish grapes. Journal Kornerup A, Wanscher JH (1978). Methuene handbook of of the Science of Food and Agriculture 84: 541–546. colour. Eyre Methuen, London, UK. Cabañes FJ, Accensi F, Bragulat MR, Abarca ML, Castellá Kozakiewicz Z (1989). Aspergillus species on stored G, Minguez S, Pons A (2002). What is the source of products. Mycological Papers 161: 1–188. ochratoxin A in wine? International Journal of Food Kusters-van Someren M, Samson RA, Visser J (1991). The Microbiology 79: 213–215. use of RFLP analysis in classification of the black As- Cutler HG, Crumley FG, Cox RH, Hernandez O, Cole RJ, pergilli: reinterpretation of the Aspergillus aggregate. Dorner JW (1979). Orlandin: a nontoxic fungal metabo- Current Genetics 19: 21–26. lite with plant growth inhibiting properties. Journal of Magnoli C, Violanta M, Combina M, Palacia G, Dalcero A Agricultural and Food Chemistry 27: 592–595. (2003). Mycoflora and ochratoxin-producing strains of Dalcero A, Magnoli C, Hallak C, Chiacchiera SM, Palacio Aspergillus section Nigri in wine grapes in Argentina. G, Rosa CAR (2002) Detection of ochratoxin A in ani- Letters in Applied Microbiology 37: 179–184. mal feeds and capacity to produce this mycotoxin by Mitchell D, Parra R, Aldred D, Magan N (2004). Water and Aspergillus section Nigri in Argentina. Food Additives temperature relations of growth and ochratoxin A pro- and Contaminants 19: 1065–1072. duction by Aspergillus carbonarius strains from grapes Frisvad JC, Thrane U (1987). Standardized high- in Europe and Israel. Journal of Applied Microbiology performance liquid chromatography of 182 mycotoxins 97: 439–445. and other fungal metabolites based on alkylphenone re- Mossereay R (1934). Les Aspergillus de la section “Niger” tention indices and UV-VIS spectra (diode-array detec- Thom and Church. La Cellule 43: 203–285. tion). Journal of Chromatography 404: 1295–214. Mouchacca J (1999). A list of novel fungi described from Frisvad JC, Frank JM, Houbraken JAMP, Kuijpers AFA, the Middle east, mostly from non-soil substrata. Nova Samson RA (2004). New ochratoxin producing species Hedwigia 68: 149–174. of Aspergillus section Circumdati. Studies in Mycology Nakajima M, Tsubouchi H, Miyabe M, Ueno Y (1997). 50: 23–43. Survey of aflatoxin B1 and ochratoxin A in commercial Fujimoto Y, Miyagawa H, Tsurushima T, Irie H, Okamura green coffee beans by high-performance liquid chroma- K, Ueno T (1993). Structures of antafumicins A and B, tography linked with immunoaffinity chromatography. novel antifungal substances produced by the fungus As- Food and Agricultural Immunology 9: 77–83. pergillus niger NH-401. Bioscience Biotechnology and Ono H, Kataoka A, Koakutsu M, Tanaka K, Kawasugi S, Biochemistry 57: 1222–1224. Wakazawa M, Ueno Y, Manabe M (1995). Ochratoxin Hayashi H, Furutsuka K, Shiono Y (1999). Okaramins H A producibility by strains of Aspergillus niger group and I, new okaramine congeners, from Aspergillus acu- stored in IFO collection. Mycotoxins 41: 47–51. leatus. Journal of Natural Products 62: 315–317. Overy DP, Blunt JW (2004). Corymbiferan lactones from Heenan CN, Shaw KJ, Pitt JI (1998). Ochratoxin A produc- Penicillium hordei: stimulation of novel phenolic me- tion by Aspergillus carbonarius and A. niger isolates 59 SAMSON ET AL.

tabolites using plant tissue media. Journal of Natural Steiman R, Guiraud P, Saga L, Siegle-Murandi, F (1994). Products, in press. New strains from Israel in the Aspergillus niger group. Parenicová L, Benen JAE, Samson RA, Visser J (1997). Systematic and Applied Microbiology 17: 620–624. Evaluation of RFLP analysis of the classification of se- Suárez-Quiroz M, Gonzáles-Rios O, Barel M, Guyot B, lected black aspergilli. Mycological Research 101: 810– Schorr-Galindo S, Guiraud J-P (2004). Study of ochra- 814. toxin A-producing strains in coffee processing. Interna- Parenicová L, Skouboe P, Frisvad JC, Samson RA, Rossen tional Journal of Food Science and Technology 39: L, ter Hoor-Suykerbuyk M, Visser J (2001). Combined 501–507. molecular and biochemical approach identifies Aspergil- Swofford T (2000) PAUP*: Phylogenetic analysis using lus japonicus and Aspergillus aculeatus as two species. parsimony. version 4.0. Sinaur Associates, Sunderland, Applied and Environmental Microbiology 67: 521–527. MA, U.S.A. Peterson SW (2000). Phylogenetic relationships in Asper- Tanaka H, Wang P, Namiki M (1972). Structure of gillus based on rDNA sequence analysis. In: Integration aurasperone C. Agricultural and Biological Chemistry of modern taxonomic methods for Penicillium and As- 36: 2511–2517. pergillus systematics (Samson RA, Pitt JI, eds.), Har- Taniwaki MH, Pitt JI, Teixeira AA, Iamanaka BT (2003). wood Academic Publishers, Amsterdam, the Nether- The source of ochratoxin A in Brazilian coffee and its lands: 323–355. formation in relation to processing methods. Interna- Rai JN, Chowdhery HJ (1979). Two new species of Asper- tional Journal of Food Microbiology 82: 173–179. gillus from mangrove swamps of West Bengal, India. TePaske MR, Gloer JB, Wicklow DT, Dowd P (1989a). Kavaka 7: 17–20. Three new aflavinines from the sclerotia of Aspergillus Raper KB, Fennell DI (1965) The genus Aspergillus. tubingensis. Tetrahedron 45: 4961–4968. Williams and Wilkins, Baltimore. TePaske MR, Gloer JB, Wicklow DT, Dowd P (1989b). Rocha RCA da, Palacios V, Combina M, Fraga ME, De Tubingensin A: an antiviral carbazole alkaloid from the Oliveira Rekson A, Magnoli CF, Dalcero, A (2002). Po- sclerotia of Aspergillus tubingensis. Journal of Organic tential ochratoxin A producers from wine grapes in Ar- Chemistry 54: 4743–4746. getina and Brazil. Food Additives and Contaminants 19: TePaske MR, Gloer JB, Wicklow DT, Dowd P (1989c). 408–414. The structure of tubingensin B: a cytotoxic carbazole Sage L, Krivobok S, Delbos E, Seigle-Murandi F, Creppy alkaloid from the sclerotia of Aspergillus tubingensis. EE (2002). Fungal flora and ochratoxin A production in Tetrahedron Letters 30: 5965–5968. grapes and musts from France. Journal of Agricultural Téren J, Varga J, Hamari Z, Rinyu E, Kevei F (1996). and Food Chemistry 50: 1306–1311. Immunochemical detection of ochratoxin A in black As- Sage L, Garon D, Seigle-Murandi F (2004). Fungal micro- pergillus strains. Mycopathologia 134, 171–176. flora and ochratoxin A risk in French wineyards. Jour- Téren J, Palagyi A, Varga J (1997). Isolation of ochratoxin nal of Agricultural and Food Chemistry 52: 5764–5768. producing Aspergilli from green coffee beans of differ- Samson RA (1979). A compilation of the Aspergilli de- ent origin. Cereal Research Communications 25: 303– scribed since 1965. Studies in Mycology (Baarn) 18: 1– 304. 38. Turner WB, Aldridge DC 1983. Fungal metabolites II. Samson RA (1992). Current taxonomic schemes of the Academic Press, New York. genus Aspergillus and its teleomorphs. In Aspergillus: Ueno Y, Kawakura O, Sugiura Y, Horiguchi K, Nakajima Biology and industrial applications (Eds Bennett, J.W. M, Yamamoto K, Sato S (1991). Use of monoclonal an- & Klich, M.A.). Boston, Butterworth-Heinemann. pp. tibodies, enzyme-linked immunosorbent assay and im- 355–390. munoaffinity column chromatography to determine Samson RA, Frisvad JC (2004). Penicillium subgenus ochratoxin A in porcine sera, coffee products and toxin- Penicillium: new taxonomic schemes, mycotoxins and producing fungi. In: Castagnero, M., Plestina, R., Dir- other extrolites. Studies in Mycology (Utrecht) 49: 1– heimer, G., Chernozemsky, IN and Bartsch, H (eds.) 157. Mycotoxins, endemic nephropathy and urinary tract tu- Samson RA, Hoekstra ES, Frisvad JC (2004). Introduction mors. International Agency for Research on Cancer, to food- and airborne fungi. 7th ed. Centraalbureau voor Lyon, pp. 71–75. Schimmelcultures, Utrecht. Urbano GR, Taniwaki MR, Leitao MF de F, Vicentini MC Samson RA, Seifert KA, Kuijpers AFA, Houbraken JAMP, (2001). Occurrence of ochratoxin A-producing fungi in Frisvad JC (2004). Phylogenetic analysis of Penicillium raw Brazilian coffee. Journal of Food Protection 64: subgenus Penicillium using partial β-tubulin sequences. 1226–1230. Studies in Mycology (Utrecht) 49: 175–200. Varga J, Kevei F, Hamari Z, Tóth B, Téren J, Croft JH, Schuster E, Dunn-Coleman N, Frisvad JC, van Dijck PWM Kozakiewicz Z (2000). Genotypic and phenotypic vari- (2002). On the safety of Aspergillus niger – a review. ability among black aspergilli. In: Samson, R.A. and Applied Microbiology and Biotechnology 59: 426–435. Pitt, J.I. (eds.): Integration of modern taxonomic meth- Sings HL, Harris GH, Dombrowski AW (2001). Dihydro- ods for Penicillium and Aspergillus classification. Har- carbazole alkaloids from Aspergillus tubingensis. Jour- wood Academic Publishers, Amsterdam, pp. 397–411. nal of Natural Products 64: 836–838. Varga J, Kevei É, Rinyu E, Téren J, Kozakiewicz Z (1996). Smedsgaard J (1997). Micro-scale extraction procedure for Ochratoxin production by Aspergillus species. Applied standardized screening of fungal metabolite production and Environmental Microbiology 62, 4461–4464. in cultures. Journal of Chromatography A 760: 264– Varoglou M, Crews P (2000). Biosynthetically diverse 270. compounds from a seawater culture of sponge-derived

60 NEW SPECIES OF ASPERGILLUS SECTION NIGRI

Aspergillus niger. Journal of Natural Products 63: 41– Vries RP de, Frisvad JC, van de Vondervoort PJI, Burgers 43. K, Kuijpers AFA, Samson RA, Visser J (2004b).Asper- Varoglou M, Corbett, TH, Valeriote FA, Crews P (1997). gillus vadensis, a new species of the group of black As- Asperazine, a selective cytotoxic alkaloid from a pergilli. Antonie van Leeuwenhoek, in press. sponge-derived culture of Aspergillus niger. Journal of Organic Chemistry 62: 7078–7079. Wicklow DT, Dowd PF, Alftafta AA, Gloer JB (1996). Vleggaar R, Steyn PS, Nagel DW (1974). Constitution and Ochratoxin A: an antiinsectan metabolite from the scle- abosolute configuration of austdiol, the main toxic me- rotia of Aspergillus carbonarius NRRL 369. Canadian tabolite of Aspergillus ustus. Journal of the Chemical Journal of Microbiology 42: 1100–1103. Society Perkin Transactions I 1974: 45–49. Yokoyama K, Wang L, Miyaji M, Nishimura K (2001). Vries RP de, Burgers K, van de Vondervoort PJI, Frisvad Identification, classification and phylogeny of the As- JC, Samson RA, Visser J (2004a). A new black Asper- pergillus section Nigri inferred from mitochondrial cy- gillus species, A. vadensis, is a promising host for ho- tochrome b gene. FEMS Microbiology Letters 200: 241– mologous and heterologous protein production. Applied 246. and Environmental Microbiology 70: 3954–3959.